The present invention relates to an inhibitor that can be deactivated by a reagent produced by a target cell and to its preparation and use. The inhibitor can be administered alone or together with an active agent such that the activity or toxicity of the active agent is reduced until it reaches a target cell producing a reagent wherein the inhibitor is cleaved by said reagent. Specific cleavage of the inhibitor causes inhibitory activity reduction of the inhibitor and as a consequence activity of the active agent is restored.
In order to improve therapeutic efficacy, the local concentration of biologically active agents in a disease site has to be increased without causing severe side effects. One way to decrease toxicity is to inject low doses of an active agent that are effective against cancer directly to tumor. The introduction of cytokine genes into tumor cells and subsequent local secretion can also circumvent the systemic cytokine toxicity. Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to generate high local concentrations of cytokines in the tumor microenvironment is to use fusion proteins. Cytokine can also be delivered to target tumor cells using tumor specific antibodies. Effective local cytokine concentrations can be achieved at tumor sites. Immunoglobulin and cytokine can be fused into one protein molecule. The engineered antibody-cytokine fusion proteins combine the targeting ability of tumor-specific antibody with the activity of cytokines. These fusion proteins target cytokine to tumors, stimulate immune destruction of tumors and limit severe toxic side-effects of high dose cytokine administration. Monoclonal antibodies can specifically bind to a receptor or other target on the surface of disease cells and produce a desired therapeutic effect. These surface proteins are usually overexpressed on cancer cells and also expressed, to some degree, on normal cells. Such expression pattern creates difficulties in the tumor-selective antibody delivery and decreases specificity of the therapy. Higher amounts of antibodies are required to achieve the desired effects and result in increased side effects and higher cost of treatment.
Angiogenesis and cancer invasion are linked to the overexpression of proteases namely matrix metalloproteinases (MMPs) and prostate specific antigen (PSA). Matrix metalloproteinases belong to the group of endogenous proteases which are able to degrade various components of extracellular matrix, such as collagen, elastin and gelatin. A positive correlation between MMP expression and increased invasiveness and progression has been demonstrated in a wide range of human cancers including breast, colon, lung and prostate. The tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis as well. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors.
The cancer-specific proteases have been used to activate anti-cancer compounds at the tumor site. Staphylococcal toxin was constructed by fusion with a peptide that inactivated the protein. This constructs was highly susceptible to peptide cleavage by cathepsin B, a protease secreted by certain metastatic tumor cells. The toxin obtained by this cleavage was rendered active and permeabilized specifically malignant cells [Panchal, 1996].
Target-specific retroviral vectors have been developed that use the cancer-specific proteases for their activation. Retroviral vectors for selective gene delivery were targeted to matrix metalloprotease expressing cells. Infectivity of viral vector was blocked by a polypeptide fused to the viral envelope glycoprotein. In the presence of exogenous MMPs produced by target cells, the envelope function was restored when a protease cleaved the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity was achieved by engineering the sequence of the linker. In vivo, the targeted vectors showed strong selectivity for matrix metalloprotease-rich tumor xenografts [Peng, 1997; Peng, 1999]. A single-chain variable fragment antibody (scFv) directed against the surface melanoma glycoprotein was fused to the amphotropic murine leukemia virus envelope to target this retroviruses specifically to melanomas. A peptide containing matrix metalloprotease (MMP) cleavage site linked the antibody with the envelope. Following virus attachment to target cell, MMP cleaved the peptide, the antibody was removed and allowed virus infection. This approach produced efficient, targeted retroviruses suitable for in vivo gene delivery [Martin, 1999; Martin, 2002]. A strategy to target cytotoxic agents specifically to sites of metastatic cancer that secretes proteases has been developed. Doxorubicin was linked to the carrier peptide moiety substantially inhibiting the non-specific toxicity of the drug. The carrier, PSA-specific peptide, determines target specificity of the drug. The drug becomes activated when processed proteolytically within prostate cancer metastases by prostate-specific antigen. PSA, which is secreted by prostatic glandular cells cleaves specifically the peptide part of the prodrug activating the therapeutic drugs and exerting their toxicity. This strategy can be used to deliver higher intratumoral levels of doxorubicin [Denmeade, 1998; DeFeo-Jones, 2000; Khan, 2000; Denmeade, 2001; Wong, 2001; DiPaola, 2002; Jones, 2002; Mhaka, 2002].    U.S. Pat. No. 6,080,575 discloses a substance which is activated by an enzyme which is released from mammalian cells.    U.S. Pat. No. 6,593,132 discloses a protein having an A chain of a ricin-like toxin, a B chain of a ricin like toxin and a heterologous linker amino acid sequence, linking the A and B chains.    U.S. Pat. No. 6,423,513 discloses a protease-activatable Pseudomonas exotoxin A-like proproteins and methods of using these proproteins for killing target cells.    U.S. Pat. No. 5,599,686 discloses oligopeptides which comprise amino acid sequences that are recognized and proteolytically cleaved by free prostate specific antigen.    U.S. Pat. No. 5,976,535 discloses pretargeting protocols for the enhanced localization of cytotoxins to target sites and cytotoxic combinations. The application teaches targeting of cytotoxins using biotin-streptavidin conjugates.    U.S. Pat. No. 6,036,955 discloses specific in vivo coagulation of tumor vasculature, causing tumor regression, through the site-specific delivery of a coagulant using a bispecific antibody.    U.S. Pat. No. 5,098,702 discloses administration to the mammalian host a synergistically effective amount of TNF and IL-2 or of TNF and IFN-beta, or of TNF, IL-2 and IFN-beta in combination.    U.S. Pat. No. 5,078,996 discloses a treatment by direct administration of therapeutically effective quantities of activated granulocyte-macrophage colony stimulating factor.    U.S. Pat. No. 5,447,851 discloses a specifically cleavable linker peptide functionally interposed between the cytokine receptor polypeptide and the IgG heavy chain polypeptide. Such a linker peptide provides by its inclusion in the chimeric construct, a site within the resulting chimeric polypeptide which may be cleaved in a manner to separate the active cytokine receptor polypeptide from the intact IgG heavy chain polypeptide.    U.S. Pat. No. 5,856,456 discloses a peptide linker useful for connecting polypeptide constituents into a novel linked fusion polypeptide.    DeFeo-Jones, D., Garsky, V. M., Wong, B., Feng, D. M., Bolyar, T., Haskell, K., Kiefer, D. M., Leander, K., McAvoy, E., Lumma, P., Wai, J., Senderak, E. T., Motzel, S. L., Keenan, K., Van Zwieten, M., Lin, J. H., Freidinger, R., Huff, J., Oliff, A. and Jones, R. E. (2000) A peptide-doxorubicin ‘prodrug’ activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo. Nat Med, 6, 1248-1252.    Denmeade, S. R., Nagy, A., Gao, J., Lilja, H., Schally, A. V. and Isaacs, J. T. (1998) Enzymatic activation of a doxorubicin-peptide prodrug by prostate-specific antigen. Cancer Res, 58, 2537-2540.    Denmeade, S. R., Sokoll, L. J., Chan, D. W., Khan, S. R. and Isaacs, J. T. (2001) Concentration of enzymatically active prostate-specific antigen (PSA) in the extracellular fluid of primary human prostate cancers and human prostate cancer xenograft models. Prostate, 48, 1-6.    DiPaola, R., Rinehart, J., Nemunaitis, J., Ebbinghaus, S., Rubin, E., Capanna, T., Ciardella, M., Doyle Lindrud, S., Goodwin, S., Fontaine, M., Adams, N., Williams, A., Schwartz, M., Winchell, G., Wickersham, K., Deutsch, P. and Yao, S. L. (2002) Characterization of a novel prostate-specific antigen-activated peptide-doxorubicin conjugate in patients with prostate cancer. J Clin Oncol, 20, 1874-1879.    Jones, G. B., Mitchell, M. O., Weinberg, J. S., D'Amico, A. V. and Bubley, G. J. (2002) Towards enzyme activated antiprostatic agents. Bioorg Med Chem Lett, 10, 1987-1989.    Khan, S. R. and Denmeade, S. R. (2000) In vivo activity of a PSA-activated doxorubicin prodrug against PSA-producing human prostate cancer xenografts. Prostate, 45, 80-83.    Martin, F., Chowdhury, S., Neil, S., Phillipps, N. and Collins, M. K. (2002) Envelope-targeted retrovirus vectors transduce melanoma xenografts but not spleen or liver. Mol Ther, 5, 269-274.    Martin, F., Neil, S., Kupsch, J., Maurice, M., Cosset, F. L. and Collins, M. (1999) Retrovirus Targeting by Tropism Restriction to Melanoma Cells. J Virol, 73, 6923-6929.    Mhaka, A., Denmeade, S., Yao, W., Isaacs, J. and Khan, S. (2002) A 5-fluorodeoxyuridine prodrug as targeted therapy for prostate cancer. Bioorg Med Chem Lett, 12, 2459.    Panchal, R. G., Cusack, E., Cheley, S. and Bayley, H. (1996) Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat Biotechnol, 14, 852-856.    Park, S. H. and Raines, R. T. (2000) Genetic selection for dissociative inhibitors of designated protein-protein interactions. Nature Biotechnology, 18, 847-851.    Peng, K. W., Morling, F. J., Cosset, F. L., Murphy, G. and Russell, S. J. (1997) A gene delivery system activatable by disease-associated matrix metalloproteinases. Hum Gene Ther, 8, 729-738.    Peng, K. W., Vile, R., Cosset, F. L. and Russell, S. (1999) Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Ther, 6, 1552-1557.    Wong, B. K., DeFeo-Jones, D., Jones, R. E., Garsky, V. M., Feng, D. M., Oliff, A., Chiba, M., Ellis, J. D. and Lin, J. H. (2001) PSA-specific and non-PSA-specific conversion of a PSA-targeted peptide conjugate of doxorubicin to its active metabolites. Drug Metab Dispos, 29, 313-318.